The intracellular phosphorylation of proteins is critical for a plethora of regulatory and signalling pathways in eukaryotic cells. Phosphorylation events can govern a wide range of cellular processes, including cell proliferation, differentiation, transcription, and morphology. An essential component of these signalling pathways is the ability of the cell to desensitize, recycle, and counteract phosphorylation signals. The cell primarily utilizes enzymes, termed phosphatases, which remove the phosphate on tyrosine, serine, and threonine side chains. The protein phosphatases are divided into three groups according to catalytic function: (1) protein phosphatases that dephosphorylate serine and threonine residues; (2) protein phosphatases which dephosphorylate tyrosine residues; and (3) dual specificity protein phosphatases which dephosphorylate serine, threonine and tyrosine residues.
Serine/threonine protein phosphatases are associated with the regulation of cholesterol biosynthesis, glycogen metabolism, muscle contractility, calcium ion channels, protein synthesis, regulation of the G2 to M transition of the cell cycle, regulation of glycolysis (6-phosphofructo-2-kinase and pyruvate kinase), glycogenolysis (phosphorylase kinase subunit), gluconeogenesis (fructose-2,6-bisphosphatase and pyruvate kinase), amino-acid degradation (phenylalanine hydroxylase), lipid metabolism (acetyl-CoA carboxylase), catecholamine synthesis (tyrosine hydroxylase) and protein synthesis (elongation factor 2).
Protein tyrosine phosphatases (PTPs) are a family of intracellular and integral membrane phosphatases that dephosphorylate tyrosine residues in proteins. PTPs have been identified in mammals, Drosophila and Schiz. pombe and are implicated in the control of normal and neoplastic growth and proliferation. They have also been found encoded by plasmids in bacteria of the genus Yersinia, where they are implicated in pathogenicity.
Dual specificity phosphatases hydrolyze phosphotyrosine, phosphothreonine, and phosphoserine residues (for a review, see, e.g., Fauman and Saper (1996) Trends in Biochem. 21:412). This class of proteins is exemplified by the VH1 or vaccinia virus late HI gene protein, whose catalytic activity is required for vaccinia virus replication. A human homolog of VH1, VHR, has also been identified. VH1-like dual specificity phosphatase can also include the phosphatases PAC-1 and CL100/MKP-1, hVH-2/MKP-2, hVH-3, MKP-3, MKP-X, MKP-4, hVH-5, and M3/6 proteins. The PAC-1 and CL100 proteins hydrolyze phosphothreonine and phosphotyrosine residues on phosphorylated MAP (mitogen activated protein) kinases. In order to modulate signalling events, the activity and expression of dual specificity phosphatases can be finely regulated. For example, the PAC-1 and CL100 phosphatase can be induced by growth factors (Keyse, S (1995) Biochim. Biophys. Acta1265:152-160).
Thus, the function of dual specificity phosphatase proteins can be critical for the regulation of cellular processes such as proliferation and differentiation. Given the important biological roles and properties of phosphatases, there exists a need for the identification of novel genes encoding such proteins as well as for the discovery of modulators of such molecules for use in regulating a variety of normal and/or pathological cellular processes.
The present invention is based, in part, on the discovery of novel dual specificity phosphatases, referred to herein as xe2x80x9c21117xe2x80x9d or xe2x80x9c38692xe2x80x9d nucleic acid and protein molecules. The nucleotide sequence of cDNAs encoding 21117 and 38692 are shown in SEQ ID NOs:1 and 4, respectively and the amino acid sequences of 21117 and 38692 polypeptides are shown in SEQ ID NOs:2 and 5, respectively. In addition, the nucleotide sequence of the 21117 and 38692 coding regions are depicted in SEQ ID NOs:3 and 6, respectively.
Accordingly, in one aspect, the invention features a nucleic acid molecule that encodes a 21117 or 38692 protein or polypeptide, e.g., a biologically active portion of the 21117 or 38692 protein. In a preferred embodiment, the isolated nucleic acid molecule encodes a polypeptide having the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:5. In other embodiments, the invention provides isolated 21117 or 38692 nucleic acid molecules having the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:6. In still other embodiments, the invention provides nucleic acid molecules that are substantially identical (e.g., naturally occurring allelic variants) to the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:6. In other embodiments, the invention provides a nucleic acid molecule that hybridizes under a stringent hybridization condition as described herein to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, 3, 4, or 6, wherein the nucleic acid encodes a full length 21117 or 38692 protein or an active fragment thereof.
In a related aspect, the invention further provides nucleic acid constructs that include the 21117 or 38692 nucleic acid molecules described herein. In certain embodiments, the nucleic acid molecules of the invention are operatively linked to native or heterologous regulatory sequences. Also included are vectors and host cells containing the 21117 or 38692 nucleic acid molecules of the invention, e.g., vectors and host cells suitable for producing 21117 or 38692 nucleic acid molecules and polypeptides.
In another related aspect, the invention provides nucleic acid fragments suitable as primers or hybridization probes for the detection of 21117 or 38692-encoding nucleic acids.
In a preferred embodiment, a nucleic acid fragment includes at least one, two and preferably more, nucleotides from the sequence of nucleotide 1 to 2985 of SEQ ID NO:1.
In a preferred embodiment, a nucleic acid fragment includes at least one, preferably more, nucleotides from the sequence of nucleotides 1 to 432 of SEQ ID NO:4, or nucleotides 850 to 1114 of SEQ ID NO:4.
In still another related aspect, isolated nucleic acid molecules that are antisense to a 21117 or 38692 encoding nucleic acid molecule are provided.
In another aspect, the invention features 21117 or 38692 polypeptides and biologically active or antigenic fragments thereof that are useful, e.g., as reagents or targets in assays applicable to treatment and diagnosis of 21117 or 38692 mediated or related disorders, e.g., liver or hematopoietic cell associated disorders. In another embodiment, the invention provides 21117 or 38692 polypeptides having a 21117 or 38692 activity. Preferred polypeptides are 21117 or 38692 proteins including at least one dual specificity phosphatase catalytic domain, and, preferably, having a 21117 or 38692 activity, e.g., a 21117 or 38692 activity as described herein.
In other embodiments, the invention provides 21117 or 38692 polypeptides, e.g., a 21117 or 38692 polypeptide having the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:5; an amino acid sequence that is substantially identical to the amino acid sequence shown in SEQ ID NOs:2 or 5; or an amino acid sequence encoded by a nucleic acid molecule having a nucleotide sequence that hybridizes under a stringent hybridization condition as described herein to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOs:1, 3, 4, 6, wherein the nucleic acid encodes a full length 21117 or 38692 protein or an active fragment thereof.
In a related aspect, the invention provides 21117 or 38692 polypeptides or fragments operatively linked to non-21117 or 38692 polypeptides to form fusion proteins.
In another aspect, the invention features antibodies and antigen-binding fragments thereof, that react with, or more preferably, specifically bind 21117 or 38692 polypeptides.
In another aspect, the invention provides methods of screening for compounds that modulate the expression or activity of the 21117 or 38692 polypeptides or nucleic acids.
In still another aspect, the invention provides a process for modulating 21117 or 38692 polypeptide or nucleic acid expression or activity, e.g. using the screened compounds. In certain embodiments, the methods involve treatment of conditions related to aberrant (e.g., decreased) activity or expression of the 21117 or 38692 polypeptides or nucleic acids, such as conditions involving aberrant cellular proliferation of a 21117- or 38692-expressing cell, e.g., a breast, colon, lung, or adipose cell, a liver cell, bone, endothelial cell, or a hematopoeitic cell (e.g., a myeloid (neutrophil) cell, a monocyte, an erythroid cell, a bone marrow cell, a CD34-expressing cell, a megakaryocyte). The condition may involve increased hematopoeitic cell activity or proliferation as in the case of leukemia, e.g., an erythroleukemia; or decreased hematopoietic cell differentiation as in the case of, e.g., an anemia.
In still another aspect, the invention features a method of modulating (e.g., enhancing or inhibiting) the proliferation, survival, migration, and/or differentiation of a cell, e.g., a 21117 or 38692-expressing cell, e.g., a breast, colon, lung, or adipose cell, a bone cell, an endothelial cell, a liver cell, or a hematopoietic cell (e.g., a myeloid (neutrophil) cell, a monocyte, an erythroid cell, a bone marrow cell, a CD34-expressing cell, a megakaryocyte). The method includes contacting the cell with an agent that modulates the activity or expression of a 21117 or 38692 polypeptide or nucleic acid, in an amount effective to modulate the proliferation, survival, migration, and/or differentiation of the cell.
In a preferred embodiment, the 21117 or 38692 polypeptide has an amino acid sequence identical to, or substantially identical to, SEQ ID NO:2 or SEQ ID NO:5. In other embodiments, the 21117 or 38692 polypeptide is a fragment of at least 15, 20, 50, 100, 150, 180, 200, or more contiguous amino acids of SEQ ID NO:2 or SEQ ID NO:5.
In a preferred embodiment, the 21117 or 38692 nucleic acid has a nucleotide sequence identical to, or substantially identical to, SEQ ID NO:1, 3, 4, or 6. In other embodiments, the 21117 or 38692 nucleic acid is a fragment of at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or more contiguous nucleotides of SEQ ID NO:1, 3, 4, or 6.
In a preferred embodiment, the agent modulates (e.g., increases or decreases) protein phosphatase activity. In other embodiments, the agent modulates (e.g., increases or decreases) expression of the 21117 or 38692 nucleic acid by, e.g., modulating transcription, mRNA stability, etc.
In preferred embodiments, the agent is a peptide, a phosphopeptide, a small molecule, e.g., a member of a combinatorial library, or an antibody, or any combination thereof. The antibody can be conjugated to a therapeutic moiety selected from the group consisting of a cytotoxin, a cytotoxic agent and a radioactive metal ion.
In additional preferred embodiments, the agent is an antisense, a ribozyme, or a triple helix molecule, or a 21117 or 38692 nucleic acid, or any combination thereof.
In a preferred embodiment, the agent is administered in combination with a cytotoxic agent.
In a preferred embodiment, the cell, e.g., the 21117 or 38692-expressing cell, is a breast, colon, bone, endothelial, liver, lung, or an adipose cell.
In a preferred embodiment, the cell, e.g., the 21117 or 38692-expressing cell, is a hematopoietic cell, e.g., a myeloid, lymphoid or erythroid cell, or a precursor cell thereof. In other preferred embodiments, the cell, e.g., the 21117 or 38692-expressing cell, is a bone marrow erythroid cell, e.g., an erythroid progenitor (e.g., a GPA(low)CD71+ cell) or a differentiated cell, e.g., an erythrocyte or a megakaryocyte.
In a preferred embodiment, the cell, e.g., the 21117 or 38692-expressing cell, is further contacted with a protein, e.g., a cytokine. Preferably, the protein is selected from the group consisting of G-CSF, GM-CSF, stem cell factor, and erythropoietin. The protein contacting step can occur before, at the same time, or after the agent is contacted. The protein contacting step can be effected in vitro or ex vivo. For example, the cell, e.g., the 21117 or 38692-expressing cell is obtained from a subject, e.g., a patient, and contacted with the protein ex vivo. The treated cell can be re-introduced into the subject. Alternatively, the protein contacting step can occur in vivo.
In a preferred embodiment, the agent and the 21117 or 38692-polypeptide or nucleic acid are contacted in vitro or ex vivo.
In a preferred embodiment, the contacting step is effected in vivo in a subject, e.g., as part of a therapeutic or prophylactic protocol. Preferably, the subject is a human, e.g., a patient with a hematopoietic disorder or an erythroid-associated disorder. In other embodiments, the subject is a non-human animal, e.g., an experimental animal.
The contacting step(s) can be repeated.
In a preferred embodiment, the agent decreases the proliferation and/or enhances the differentiation of the cell, e.g., the 21117 or 38692-expressing cell (e.g., the hematopoietic cell, e.g., the myeloid (neutrophil) cell, the monocyte, the erythroid cell, the bone marrow cell, the CD34-expressing cell, or the megakaryocyte cell). Such agents can be used to treat or prevent cancers, e.g., leukemic cancers.
In a preferred embodiment, the agent increases the number of hematopoietic cells (e.g., myeloid (neutrophil) cells, monocytes, erythroid cells, bone marrow cells, CD34-expressing cells, megakaryocytes), by e.g., increasing the proliferation, survival, and/or stimulating the differentiation, of progenitor cells. Such agents can be used to treat or prevent hematopoietic or erythroid cell-associated disorders.
In another aspect, the invention features a method of modulating hematopoiesis, e.g., erythropoiesis, comprising contacting a 21117 or 38692-expressing cell hematopoietic cell (e.g., a myeloid (neutrophil) cell, a monocyte, an erythroid cell, a bone marrow cell, a CD34-expressing cell, a megakaryocyte), with an agent that increases or decreases the activity or expression of a 21117 or 38692 polypeptide, e.g., a polypeptide as described herein, or nucleic acid, e.g., a nucleic acid as described herein, thereby modulating the differentiation of the hematopoietic cell.
In a preferred embodiment, the agent modulates (e.g., increases or decreases) protein phosphatase activity.
In preferred embodiments, the agent is a peptide, a phosphopeptide, a small molecule, e.g., a member of a combinatorial library, or an antibody, or any combination thereof. The antibody can be conjugated to a therapeutic moiety selected from the group consisting of a cytotoxin, a cytotoxic agent and a radioactive metal ion.
In additional preferred embodiments, the agent is an antisense, a ribozyme, or a triple helix molecule, or a 21117 or 38692 nucleic acid, or any combination thereof. In another embodiment, the agent is administered in combination with a cytotoxic agent.
In a preferred embodiment, the hematopoeitic cell is a bone marrow cell, e.g., a CD34-expressing cell, an erythroid cell, e.g., an erythroid progenitor or differentiated cell, e.g., an erythrocyte or a megakaryocyte; or a liver cell.
In a preferred embodiment, the agent and the 21117 or 38692-polypeptide or nucleic acid are contacted in vitro or ex vivo.
In a preferred embodiment, the contacting step is effected in vivo in a subject, e.g., as part of a therapeutic or prophylactic protocol. In one embodiment, the subject is a patient at risk, or having a disording involving aberrant activity of a cell or tissue where a 21117- or 38692 molecule is expressed. For example, the subject is a human, e.g., a patient with a hematopoietic disorder or an erythroid-associated disorder. Alternatively, the subject can be a cancer patient, e.g., a patient with leukemic cancer. In other embodiments, the subject is a non-human animal, e.g., an experimental animal.
In a preferred embodiment, the method further includes contacting of the erythroid cell with a protein, e.g., a cytokine. Preferably, the protein is selected from the group consisting of G-CSF, GM-CSF, stem cell factor, and erythropoietin. The protein contacting step can occur before, at the same time, or after the agent is contacted. The protein contacting step can be effected in vitro or ex vivo. For example, the cell, e.g., the erythroid cell can be obtained from a subject, e.g., a patient, and contacted with the protein ex vivo. The treated cell can be re-introduced into the subject. Alternatively, the protein contacting step can occur in vivo.
The contacting step(s) can be repeated.
In a preferred embodiment, the agent increases the number of hematopoietic cells, e.g., erythroid cells, by e.g., increasing the proliferation, survival, and/or stimulating the differentiation, of hematopoietic (e.g., eythroid) progenitor cells, in the subject.
In yet another aspect, the invention features a method of treating or preventing a disorder involving aberrant expression or activity of a 21117- or a 38692 nucleic acid or polypeptide, in a subject. The method includes administering to the subject an effective amount of an agent that modulates the activity or expression of a 21117 or 38692 polypeptide or nucleic acid such that the disorder is ameliorated or prevented.
In a preferred embodiment, the 21117 or 38692 polypeptide has an amino acid sequence identical to, or substantially identical to, SEQ ID NO:2 or SEQ ID NO:5. In other embodiments, the 21117 or 38692 polypeptide is a fragment of at least 15, 20, 50, 100, 150, 180, 200, or more contiguous amino acids of SEQ ID NO:2 or SEQ ID NO:5.
In a preferred embodiment, the 21117 or 38692 nucleic acid has a nucleotide sequence identical to, or substantially identical to, SEQ ID NO:1, 3, 4, or 6. In other embodiments, the 21117 or 38692 nucleic acid is a fragment of at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or more contiguous nucleotides of SEQ ID NO:1, 3, 4, or 6.
In a preferred embodiment, the agent modulates (e.g., increases or decreases) protein phosphatase activity.
In a preferred embodiment, the agent modulates (e.g., increases or decreases) expression of the 21117 or 38692 nucleic acid by, e.g., modulating transcription, mRNA stability, etc.
In preferred embodiments, the agent is a peptide, a phosphopeptide, a small molecule, e.g., a member of a combinatorial library, or an antibody, or any combination thereof. The antibody can be conjugated to a therapeutic moiety selected from the group consisting of a cytotoxin, a cytotoxic agent and a radioactive metal ion.
In additional preferred embodiments, the agent is an antisense, a ribozyme, or a triple helix molecule, or a 21117 or 38692 nucleic acid, or any combination thereof.
In a preferred embodiment, the agent is administered in combination with a cytotoxic agent.
In a preferred embodiment, the subject is a human, e.g., a patient with a immune or a cancer disorder. In other embodiments, the subject is a non-human animal, e.g., an experimental animal.
In a preferred embodiment, the disorder is a disorder associated with aberrant activity of a cell in which a 21117 or 38692 molecule is expressed, e.g., a disorder involving aberrant activity or expression of a cell e.g., a breast, colon, lung, or adipose cell, a liver cell, bone, endothelial cell, or a hematopoeitic cell (e.g., a myeloid (neutrophil) cell, a monocyte, an erythroid cell, a bone marrow cell, a CD34-expressing cell, a megakaryocyte).
In a preferred embodiment, the disorder is a hematopoietic disorder or an erythroid-associated disorder.
In a preferred embodiment, the disorder is a cancer, e.g., leukemic cancer, a carcinoma, sarcoma or a metastatic cancer.
In a preferred embodiment, the agent decreases the proliferation and/or enhances the differentiation of a cell, e.g., a 21117 or 38692-expressing cell, e.g., a hematopoeitic cell (e.g., a myeloid (neutrophil) cell, a monocyte, an erythroid cell, a bone marrow cell, a CD34-expressing cell, a megakaryocyte), in the subject. Such agents can be used to treat or prevent cancers, e.g., leukemic cancers such as erythroid leukemias, or carcinomas, sarcomas or metastatic cancers.
In a preferred embodiment, the agent increases the number of a 21117 or 38692-expressing cells, e.g., hematopoietic cells (e.g., erythroid, myeloid, monocyte, or megakaryocyte cells), by e.g., increasing the proliferation, and/or stimulating the differentiation, of progenitor cells, in the subject.
In a preferred embodiment, the method further includes administering an effective amount of a protein, e.g., a cytokine. Preferably, the protein is selected from the group consisting of G-CSF, GM-CSF, stem cell factor, and erythropoietin to the subject. Preferably, the protein is erythropoietin. The protein can be administered before, at the same time or after, administration of the agent.
The administration of the agent and/or protein can be repeated.
In still another aspect, the invention features a method for evaluating the efficacy of a treatment of a disorder, in a subject. The method includes treating a subject with a protocol under evaluation; assessing the expression of a 21117 or 38692 nucleic acid or 21117 or 38692 polypeptide, such that a change in the level of 21117 or 38692 nucleic acid or 21117 or 38692 polypeptide after treatment, relative to the level before treatment, is indicative of the efficacy of the treatment of the disorder.
In a preferred embodiment, the subject is a human. In other embodiments, the subject is an experimental animal, e.g., an animal model for a hematopoietic- (e.g., an erythroid-) associated disorder, a cancer disorder, or an endothelial cell disorder.
In a preferred embodiment, the method can further include treating the subject with a protein, e.g., a cytokine. Preferably, the protein is selected from the group consisting of G-CSF, GM-CSF, stem cell factor, and erythropoietin prior to assessing expression levels. Preferably, the protein is erythropoietin.
The invention also features a method of diagnosing, or staging, a disorder involving aberrant activity or expression of a 21117 or 38692 nucleic acid or a 21117 or 38692 polypeptide. The method includes evaluating the expression or activity of a 21117 or 38692 nucleic acid or a 21117 or 38692 polypeptide, such that, a difference in the level of 21117 or 38692 nucleic acid or 21117 or 38692 polypeptide relative to a normal subject or a cohort of normal subjects is indicative of the disorder.
In a preferred embodiment, the subject is a human.
In a preferred embodiment, the evaluating step occurs in vitro or ex vivo. For example, a sample, e.g., a blood sample, a biopsy, is obtained from the subject.
In a preferred embodiment, the evaluating step occurs in vivo. For example, by administering to the subject a detectably labeled agent that interacts with the 21117 or 38692 nucleic acid or polypeptide, such that a signal is generated relative to the level of activity or expression of the 21117 or 38692 nucleic acid or polypeptide.
In a preferred embodiment, the disorder is a cancer, or a hematopoietic disorder, e.g., a hematopoietic or an erythroid associated disorder, as described herein.
The invention also provides assays for determining the activity of or the presence or absence of 21117 or 38692 polypeptides or nucleic acid molecules in a biological sample, including for disease diagnosis.
In further aspect, the invention provides assays for determining the presence or absence of a genetic alteration in a 21117 or 38692 polypeptide or nucleic acid molecule, including for disease diagnosis.
In yet another aspect, the invention features a method for identifying an agent, e.g., a compound, which modulates the activity of a 21117 or 38692 polypeptide, e.g., a 21117 or 38692 polypeptide as described herein, or the expression of a 21117 or 38692 nucleic acid, e.g., a 21117 or 38692 nucleic acid as described herein, including contacting the 21117 or 38692 polypeptide or nucleic acid with a test agent (e.g., a test compound); and determining the effect of the test compound on the activity of the polypeptide or nucleic acid to thereby identify a compound which modulates the activity of the polypeptide or nucleic acid.
In a preferred embodiment, the activity of the 21117 or 38692 polypeptide is protein phosphatase activity.
In a preferred embodiment, the activity of the 21117 or 38692 polypeptide is proliferation, differentiation, migration, and/or survival of a cell, e.g., a 21117 or 38692-expressing cell, e.g., a breast, colon, lung, or adipose cell, a bone cell, an endothelial cell, a liver cell, or a hematopoietic cell (e.g., a myeloid (neutrophil) cell, a monocyte, an erythroid cell, a bone marrow cell, a CD34-expressing cell, a megakaryocyte).
In preferred embodiments, the agent is a peptide, a phosphopeptide, a small molecule, e.g., a member of a combinatorial library, or an antibody, or any combination thereof.
In additional preferred embodiments, the agent is an antisense, a ribozyme, or a triple helix molecule, or a 21117 or 38692 nucleic acid, or any combination thereof.
In another aspect, the invention features a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality, and each address of the plurality having a unique capture probe, e.g., a nucleic acid or peptide sequence. At least one address of the plurality has a capture probe that recognizes a 21117 or 38692 molecule. In one embodiment, the capture probe is a nucleic acid, e.g., a probe complementary to a 21117 or 38692 nucleic acid sequence. In another embodiment, the capture probe is a polypeptide, e.g., an antibody specific for 21117 or 38692 polypeptides. Also featured is a method of analyzing a sample by contacting the sample to the aforementioned array and detecting binding of the sample to the array.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.